Host-Gated Enzymatic Release (H-GER) Enables Colorimetric Transduction for Enzyme Measurement

We introduce Host-Gated Enzymatic Release (H-GER) as an alternative colorimetric signal transduction mechanism for measuring amylase activity. This assay uses a visually colored complex formed when hydroxypropyl-γ-cyclodextrin (HP-γ-CD) binds to the aggregachromic dye CRANAD-2, with the HP side chains playing a key role in the complexation. The analytical capability of this visually addressable assay relies on changes in dye dispersity, triggered by the enzymatic release of gated CRANAD-2 from HP-γ-CD host. Upon cleavage of HP-γ-CD, the freed dye clusters in the aqueous environment, resulting in a sequence of color changes observed by the naked eye. The H-GER assay demonstrated a limit of detection of 154 U/mL for α-amylase. Analysis based on Michaelis-Menten kinetics and molecular dynamics simulations revealed that the H-GER assay exhibits good enzymatic specificity, despite showing reduced catalytic efficiency. These results demonstrate that H-GER is an effective and potentially valuable signal transduction mechanism that expands the current toolbox for developing <i>in vitro</i> colorimetric assays targeting specific enzymatic activities.